RESULTS AND DISCUSSION
Isolation and Identification of DAQH (1) and DAQJ (2).
Screening of our actinomycete secondary metabolite library
against M. tuberculosis in the microplate Alamar Blue assay
(MABA) and low-oxygen-recovery assay (LORA) led to the
selection of Lake Michigan-derived strain B026 for further
chemical investigation. A 28 L fermentation of B026 was
performed, and following the extraction of secondary
metabolites from the fermentation broth and several chromatographic
steps using bioassay-guided fractionation, 0.3 mg each
of 1 and 2 was purified using RP-C18 semipreparative HPLC
(2.4 mL min−1, gradient of 50% aqueous ACN to 100% ACN
for 25 min, followed by an isocratic flow of 100% ACN for 15
min; tR 18.6 and 22.0 min, respectively).
Detailed structure elucidation analysis for compounds 1 and
2, including full 1H and 13C NMR assignments in addition to
2D NMR data and MS experiments, is located in the
Supporting Information. Combined NMR and high-resolution
IT-TOF mass spectrometry (MS) experiments of 1 established
the molecular formula as C22H26N2O4, whereas the UV
spectrum displayed a characteristic absorption profile of
molecules from the diazaquinomycin class, thus helping to
confirm the fused diaza-anthracene core skeleton. 1H NMR
resonances for aromatic (H3, H6) and aliphatic (H11, H12)
hydrogens and their associated correlations from a heteronuclear
multiple-bond correlation (HMBC) NMR experiment
suggested that each lactam ring contained α-unsaturated, β-
alkylated carbonyl moieties (Table S1). 1H NMR data
suggested there was a clear asymmetry to the ring system,
and further exploration of the HMBC and MS data confirmed
the presence of methyl and isononyl β-substituents on either
lactam ring. The connectivity of each set of hydrogens in the
isononyl group was determined using correlation NMR
spectroscopy (COSY) and a series of one-dimensional selective
total correlation spectroscopy (TOCSY) experiments (Figure
2). Therefore, the structure of 1 is as shown (Figure 1).
Structure elucidation of DAQJ was executed in the same
fashion as DAQH, using a similar series of MS and one- and
two-dimensional NMR experiments to identify the molecule. A
14 Da increase in molecular weight and an extra resonance in
the 1H and 13C spectra suggested an additional methylene was
present in the aliphatic side chain.